Shotgun Sequencing
Shotgun sequencing is a method of sequencing long strands of DNA that are unable to be sequenced by traditional chain-termination sequencing. This is done by breaking up the DNA into smaller segments and sequencing the shorter strands in a repetitive manner which creates overlapping sequences. Computer programs are then used to align the sequences and reconstruct the whole genome. http://en.wikipedia.org/wiki/Shotgun_sequencing Methods of Shotgun Sequencing There are two main methods of shotgun sequencing: Clone-by-Clone shotgun sequencing and whole genome shotgun sequencing. In clone-by-clone sequencing, bacterial or yeast vector is used to code segments of a whole genome or a region of interest. These are called bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Contigs of BACs or YACs are then generated by using restriction enzyme site mapping to create overlapping regions in the genome. Subclones are chosen based on quality overlaps. The DNA is then purified, sheared and subcloned into a plasmid or M-13 based vector. Once cloned, random reads are created in both directions and can be used for assembly. Overlapping sequences allow whole regions of the genome to be sequenced by subsequent computational alignment. Yeast vectors can clone upwards of a megabase of DNA while bacterial vectors have the ability to clone around 100 - 200 base pairs. The most famous use of the clone-by-clone sequencing method was in the sequencing of the human genome by the Human Genome Project. The second method of shotgun sequencing is whole genome shotgun sequencing. In this method, whole genetic sequences of a particular organism are sheared and cloned into the appropriate vector. Reads are generated from both insert ends from the clones creating a highly redundant sequence and increased coverage. Finally, the sequences are then computationally aligned. This method bypasses the use of artificial chromosomes and is useful for sequencing smaller chromosomes which are less repetitive. This method was used by Craig Venter and Celera Genomics in sequencing the human genome. Shotgun Sequencing and Next-Generation Sequencing Traditional shotgun sequencing is based on Sanger sequencing method. Shotgun sequencing is now based on next-generation sequencing method. This new method generates shorter reads, but it could produce millions of them in a relatively short time. As a result, the next-generation sequencing method leads to high coverage, and saves time, therefore makes it vastly superior than Sanger sequencing method. Whole Genome Shotgun Sequencing of the Sargasso Sea In 2003, surface water samples were taken from the Sargasso Sea, a region in the middle of the North Atlantic Ocean bound by the Gulf Stream to the West and North Atlantic Current on the North. It is also bound by the Canary Current to the East and the North Atlantic Equatorial current. Due to the geography of this particular region of the ocean, a gyre is created where refuse and marine plants are deposited. Also, due to its unique environment, researchers sought to characterize its microbiome for new microbial species and genes. This was done using Whole Genome Shotgun Sequencing of the water samples collected in 2003. 1.66 million reads were collected from the samples, pooled and assembled. Using this approach 1,214,207 genes were identified with a total of 69,901 novel genes. Also greater than 60,000 16S rRNA sequences were identified illustrating prokaryotic diversity. Depending on the percentage of sequence similarity the researchers used, either 97% or 99% a total number of 143 and 643 new phylotypes were discovered respectively. This study showed that whole genome shotgun sequencing can not only be used to identify the genetic sequence of a single species but could be used in metagenomics studies to identify multiple species. Reference 1. International Human Genome Sequencing Consortium. Initial Sequencing and Analysis of the Human Genome. Nature. v. 409 no.6822. http://www.nature.com/nature/journal/v409/n6822/pdf/409860a0.pdf 2. Venter, C.J. et. al. The Sequence of the Human Genome. Science. 16 February 2001. Vol. 291 no. 5507. pp 1304-1351. http://www.sciencemag.org/content/291/5507/1304.full 3. Green, Eric D. Strategies for the systematic sequencing of complex genomes. Nature Reviews Genetics. 2, 573-583. (August 2001) http://www.nature.com/nrg/journal/v2/n8/full/nrg0801_573a.html 4. Venter, C.J. ''et al. ''Environmental Genome Shotgun Sequencing of the Sargasso Sea. Science. 2 April 2004. Vol 304 No. 5667. pp 66-74. http://www.sciencemag.org/content/304/5667/66.long%7C4